This project concerns identification of the hematopoietic progenitor cell that gives rise to B1 cells, a specialized subpopulation of B cells. B1 cells are responsible for producing so-called natural antibodies that protect against microbial infection prior to the onset of adaptive immunity and appear to play a protective role in some degenerative diseases. B1 cells differ from conventional B cells in many other important ways, including stimulation of T cells, production of immunosuppressive IL-10, and skewing of pro-inflammatory T cell differentiation. In mice, B1 cells constitute a distinct lymphocyte lineage that is generated by a unique lymphopoietic progenitor, separate and apart from the progenitor that gives rise to conventional B2 cells. The applicant laboratory has now discovered the phenotypic identity of human B1 cells, as described in more detail elsewhere. With the true nature of human B1 cells in hand, the objective of this application is to identify the specific hematopoietic/lymphopoietic origin of human B1 cells, and to support or refute the hypothesis that human B1 cells, like their murine counterparts, derive from a unique progenitor. Identifying and characterizing the B1 cell progenitor holds out the promise of being able to manipulate the development, and hence modulate the number and/or function, of mature B1 cells, to enhance or diminish the special natural antibodies, and the special effects on T cells, produced by B1 cells, in health and disease, through specific B1 progenitor cell-directed therapies. To achieve this goal, several specific aims will be pursued to: 1) Establish a model system for lymphopoiesis that fosters the development of B1 cells from cord blood and/or bone marrow hematopoietic stem cells through adoptive transfer to immunocompromised animals, supplemented by in vitro studies of B1 cell development from hematopoietic stem cells co-cultured with mesenchymal cells; 2) Identify the earliest progenitor that specifically produces B1 cells and not B2 cells through sort-purification and differentiation of phenotypically defined populations from cord blood and bone marrow; and, 3) Characterize the identified progenitor that produces human B1 cells by evaluating endogenous gene expression through single cell PCR of specific transcripts and comparison with profiles of defined lymphopoietic stages, along with evaluation of the features that characterize progenitor-generated mature B1 cells, and examination of exogenous cytokine influences on B1 cell development. Understanding the origin of human B1 cells, which have only recently been identified in normal and patient populations, is critical to understanding B1 cell development and B1 cell physiology and pathophysiology, which can lead to novel strategies for therapeutic manipulation of B1 cell numbers and function to protect against infectious and other diseases.